Project summary/Abstract The premise of the proposal is that cationic antimicrobial peptides (AMPs) can be designed to enhance systemic efficacy for sepsis treatment based on the unique properties to kill bacteria regardless of multidrug resistance (MDR) as well as to attenuate inflammation mediated by endotoxin stimulation of toll like receptor (TLR) 4. This will be accomplished using a series of rationally engineered libraries of novel peptide antibiotics (PAX) to establish the distinction between structural determinants of antimicrobial potency and those of host toxicity. Sepsis or SIRS (systemic inflammatory response syndrome) is defined as a ?life-threatening organ dysfunction caused by a dysregulated host response to infection.? Sepsis-related fatality rate is approximately 30% annually, which may reach up to 50% in more severe cases. The development of multiple classes of antibiotics toward the mid-20th century initially led to a sharp decline in sepsis-related mortality. However, persistent sepsis morbidity and mortality, even with the use of effective antibiotics, is indicative of heightened immune responses and MDR. The increased frequency of MDR bacteria has created an urgent need for the development of novel classes of antibiotics with new antimicrobial mechanisms. In addition, sepsis has a complex pathophysiology that makes it difficult to treat. By mitigating stimulation of inflammation via TLR4 signaling and eliminating the causative agent (bacteria), AMPs has the potential to eradicate the problem by its root. Over the past decade we have developed novel strategies to design and characterize cationic AMPs for treatment of infections associated with MDR bacteria based on de novo engineered cationic antimicrobial peptides (eCAPs). The use of Trp substitution on the hydrophobic side results in two lead peptides (WLBU2 and WR12) that retain broad-spectrum activity in saline, acidic pH, and human plasma, indicating the specific roles of different amino acids in AMP function. Both eCAPs were effective against 89-92% of a diverse panel of ESKAPE (MDR) pathogens compared to activity against only 50% of these strains displayed by both the human AMP LL37 and colistin. WLBU2 also displays systemic efficacy in a P. aeruginosa septicemia model, which dispels the notion that AMPs ought to be used only topically to be effective. A single systemic dose of WLBU2 protects mice injected with an otherwise lethal inoculum of P. aeruginosa. However, an important concern is a narrow therapeutic index [TI = maximum tolerated dose (MTD)/minimum therapeutic dose (mTd)]) of 3-5 (mTd of 3-4mg/kg and MTD of 12-15mg/kg) of WLBU2, which will be addressed in this proposed research. We hypothesize that AMPs can be designed for application to sepsis treatment based on a rational framework for structure-function correlations by elucidating the unique contributions of the cationic and hydrophobic contents to antibacterial selectivity, endotoxin neutralization, and host toxicity. To address this hypothesis, we will examine efficacy in murine models of bacterial sepsis and cecal ligation puncture-induced polymicrobial sepsis. This basic and translational research will result in the final selection of a single effective drug for advanced pre-clinical and clinical studies for sepsis treatment.